Proteins must be localized to the correct subcellular compartment for proper function. Protein translocation machineries fulfill this essential function by mediating signal-dependent transport of protein molecules across membranes. The importance of these transport processes for cell growth and development is evident from their direct role in a number of disease states including bacterial pathogenicity and protein misfolding diseases such as cystic fibrosis. Altered protein transporter structure or perturbed trafficking is responsible for a number of leukemias and cancers. Protein transport machineries ubiquitous in bacteria but absent from humans are excellent targets for antibiotic development. The goal of the proposed research is to advance our understanding of the molecular basis of protein transport mechanisms in bacteria. The Sec and Tat machineries will be examined using single-turnover stopped-flow fluorescence and single-molecule fluorescence microscopy techniques in order to dissect individual kinetic steps of transport. The specific aims of the project are: (1) to develop a real-time fluorescence-based kinetic assay for the Escherichia coli Sec machinery; (2) to determine the Sec and Tat transport times via stopped-flow fluorescence; (3) to develop a lipid bilayer system suitable for simultaneous electrical and single-molecule fluorescence measurements; and (4) to determine the transport kinetics of single Sec and Tat substrates via single-molecule fluorescence microscopy. The results will be widely applicable to our understanding of membrane transport mechanisms and will significantly advance the field of single-molecule biophysics.